Role of guaiacol in the degradation of arylglycerol-β-guaiacyl ether by Phanerochaete chrysosporium

Abstract It was found that guaiacol and 4-ethoxy-3-methoxy-phenylglycerol were produced via different pathways and that they were not counterpart compounds to each other in the cleavage of the β - O -4 bond of 4-ethoxy-3-methoxyphenylglycerol-β-guaiacyl ether by a white-rot fungus, Phanerochaete chrysosporium .     

Metabolism of non-phenolic diarylpropane lignin substructure model compound by Coriolus versicolor

Abstract A lignin substructure model, 1-(4-ethoxy-3,5-dimethoxyphenyl)-2-(4-ethoxy-3-methoxyphenyl)-propane-1,3-diol(I), was actively metabolized by a white-rot fungus Coriolus versicolor in low nitrogen stationary cultures favouring the ligninolytic activity in the fungus. Cleavage of the dimer I between C_α and C_β of the propanoid side chain was the major degradative reaction by the fungus.     

Upregulation of BACE1 and β-Amyloid Protein Mediated by Chronic Cerebral Hypoperfusion Contributes to Cognitive Impairment and Pathogenesis of Alzheimer’s Disease

Chronic cerebral hypoperfusion (CCH) increases the risk of Alzheimer disease (AD) through several biologically plausible pathways, but the relationship between CCH and the development of AD remains uncertain. To investigate expression of APP, BACE1 and Aβ in the hippocampus of BCCAO rats and study pathophysiological mechanism of AD from CCH. CCH rat model was established by chronic bilateral common carotid artery occlusion (BCCAO). Behavior was evaluated after BCCAO with Morris water maze and open-field task. Expression of Aβ was measured by enzyme linked immunosorbent assay (ELISA). β-Amyloid precursor protein cleavage enzyme 1 (BACE1) and β-amyloid precursor protein (APP) were tested by ELISA, Western blotting and RT-PCR. Cognitive impairment occurred with CCH by Morris water maze test and open-field task. The BACE1 and Aβ level in BCCAO rats was more increased than sham-operation control rats (P < 0.01) but APP had no difference(P > 0.05). The expression of BACE1 and Aβ has no inter-grouop difference in BCCAO rats (P > 0.05). The level of BACE1 and Aβ had positive correlation with cognitive impairment (P < 0.01) while no correlation was observed between APP and cognitive impairment. Chronic cerebral ischemia contributes to cognitive impairment and vascular pathogenesis of Alzheimer’s disease that chronic cerebral hypoperfusion increases BACE1 and Aβ level in brain.     

Microbial degradation of natural and of new synthetic polymers

Abstract In landfills, deposited waste material is usually faced with strictly anoxic conditions. This means that the design of new biodegradable polymers must take into consideration that degradation should be possible especially in the absence of molecular oxygen. Poly-β-hydroxybutyrate is depolymerized by the anaerobic fermenting bacterium Ilyobacter delafieldii through an extracellular hydrolase. Monomers are degraded inside the cells through classical β-oxidation. Polyalkanoates containing odd-numbered or branched-chain acid monomers should he degraded in an analogous manner; in most cases the final mineralization of these residues requires special pathways. A comparison of the chemistry of natural polymer biodegration leads to the conclusion that synthetic biodegradable polymers should be designed in the future to contain linkages which can be cleaved by extracellular hydrolytic enzymes. Recent findings on aerobic and anaerobic bacterial degradation of synthetic polyethers suggest that natural evolution of new depolymerizing enzymes, perhaps from existing hydrolases, could be possible in a reasonable amount of time, provided that the monomers are likely energy sources for a broad variety of microbes.     

Expression of Cytochrome P450 Side-Chain Cleavage Enzyme and 3β-Hydroxysteroid Dehydrogenase in the Rat Central Nervous System: A Study by Polymerase Chain Reaction and In Situ Hybridization

Abstract: In examining steroid synthesis in the CNS, expression of the mRNAs encoding for cytochrome P450 side-chain cleavage enzyme (P450_SCC) and 3β-hydroxysteroid dehydrogenase/Δ⁵-Δ⁴ isomerase (3β-HSD) has been studied in the rat brain. P450_SCC transforms cholesterol into pregnenolone and 3β-HSD transforms pregnenolone into progesterone. PCR was used to amplify cDNA sequences from total RNA extracts. Classical steroidogenic tissues, like adrenal and testis, as well as the non-steroidogenic tissue lung have been used as controls. The expression of P450_SCC and 3β-HSD have been demonstrated by PCR in cortex, cerebellum, and spinal cord. In addition, primary cultures of rat cerebellar glial cells and rat cerebellar granule cells were found to express P450_SCC and 3β-HSD at comparable levels. Furthermore, three of the four known isoenzymes of 3β-HSD were identified, as determined using selective PCR primers coupled with discriminative restriction enzymes and sequencing analysis of the amplified brain products. Using RNA probes, in situ hybridization indicated that P450_SCC and 3β-HSD are expressed throughout the brain at a low level and mainly in white matter. Enrichment of glial cell cultures in oligodendrocytes, however, does not increase the relative abundance of P450_SCC and 3β-HSD mRNA detected by PCR. This discrepancy suggests that the developmental state of cultured cells and their intercellular environment may be critical for regulating the expression of these enzymes. These findings support the proposal that the brain apparently has the capacity to synthesize progesterone from cholesterol, through pregnenolone, but that the expression of these enzymes appears to be quite low. Furthermore, the identification of these messages in cerebellar granule cell cultures implies that certain neurons, in addition to glial cells, may express these steroidogenic enzymes.     

The cleavage program in the 2d cell lineage of Tubifex embryos

Early development in clitellate annelids is characterized by a highly stereotyped sequence of unequal, spiral cleavages. Cell 2d (i.e., the second micromere of the D quadrant) in the oligochaete Tubifex tubifex also undergoes an evolutionarily conserved sequence of cell division to produce four bilateral pairs of ectodermal teloblasts that act as embryonic stem cells. This study was conducted to characterize each of the 15 rounds of cell division that occur in the 2d cell lineage in this clitellate. After its occurrence, cell 2d undergoes three rounds of highly unequal divisions, giving off the first smaller daughter cell toward the posterior right of the larger daughter cell, the second cell toward the posterior left, and the third cell toward the anterior side of the cell; the larger daughter cell that results from the third division (i.e., the great-granddaughter cell of 2d) then divides equally into a bilateral pair of NOPQ proteloblasts. Cell NOPQ on either side of the embryo undergoes 11 rounds of cell division, during which ectoteloblasts N, Q, and O/P are produced in this order. After its appearance, NOPQ undergoes highly unequal divisions twice cutting off the smaller cells toward the anterior end of the embryo and then divides almost equally into ectoteloblast N and proteloblast OPQ. After its appearance, OPQ undergoes highly unequal divisions twice giving off the first smaller cell toward the anterior and the second smaller cell toward the posterior of the embryo and then divides almost equally into ectoteloblast Q and proteloblast OP. Finally, OP undergoes highly unequal division four times after its birth budding off the smaller cells toward the anterior and then cleaves equally into ectoteloblasts O and P. In the unequally dividing cells of the 2d cell lineage, the mitotic apparatus (MA), which forms at the cell's center, moves eccentrically toward the cortical site where the smaller cell will be given off. The moving MA is oriented perpendicular to the surface it approaches, and its peripheral pole becomes closely associated with the cell cortex. In contrast, the MA involved in the equal divisions remains in the cell center throughout mitosis. The key features of the cleavage program in the 2d cell lineage are discussed in light of the present observations. The mechanical aspects of unequal cleavage in the 2d cell lineage and the modes of specification of MA orientation are discussed. A comparison of the cleavage mode in the 2d cell lineage is also performed among six selected clitellate annelid species.     

蛋白质特异性断裂试剂研究进展

蛋白质特异性断裂试剂是近年来发展起来的一些具有特异性断裂肽键功能的化学工具．这些断裂试剂可分为两类,一类通过氧化断裂机制实现蛋白空间结构特异性切割,另一类通过水解断裂机制实现序列特异性切割．蛋白质特异性断裂试剂在蛋白质序列测定,蛋白质的结构与功能研究,蛋白质与核酸相互作用研究以及新型化学治疗药物的合成等方面有着广阔的应用前景．     

Glucose exerts a negative effect over a peroxidase from <Emphasis Type="Italic">Trichosporon asahii</Emphasis>, with carotenoid cleaving activity

Tobacco aroma compounds were generated via lutein cleavage by the combined action of a yeast and a bacterium identified as Trichosporon asahii and Paenibacillus amylolyticus, respectively. In this study, an inverse relationship between glucose concentration and the generation of three compounds, present in the tobacco aroma profile, was observed in mixed cultures. In order to identify the organism sensitive to the sugar effect, both were grown separately. The presence of glucose suppressed β-ionone production by T. asahii grown with lutein. However, the biotransformation of the ionone into its reduced derivatives (7,8-dihydro-β-ionone and 7,8-dihydro-β-ionol) by P. amylolyticus was not affected by the sugar . This pointed to the cleavage of lutein, a step within the process necessary for the synthesis of β-ionone, as the target of the glucose effect. In vitro studies with crude extracts and concentrated cell-free medium derived from T. asahii cultures showed that the carotenoid breakdown activity was located extracellularly and only detected in supernatants from yeast cells grown in the absence of the sugar. Rather than an inhibition or a mechanism affecting the enzyme secretion, the glucose effect on lutein degradation comprised another regulatory level. Further experiments showed that the enzyme responsible for lutein breakdown and susceptible to the sugar effect exhibited a high degree of identity to fungal peroxidases, studied as well, for their involvement in carotenoid cleavage.     

Synthesis, characterization, and C-H/C-C cleavage reactions of two rhodium-trispyrazolylborate dihydrides

Treatment of Tp′Rh(PMe₃)Cl₂ and Tp′Rh(CNCH₂CMe₃)Cl₂ with Cp₂ZrH₂ produces Tp′Rh(PMe₃)H₂ and Tp′Rh(CNCH₂CMe₃)H₂, respectively, in excellent yield. Photolysis of benzene solutions of each dihydride complex generates hydrogen and the fragment [Tp′Rh(L)] which inserts into the solvent C-H bond. The phosphine dihydride has also been shown to be a catalyst for the hydrogenation of biphenylene, showing a capability to cleave C-C bonds. Reductive elimination of benzene from Tp′Rh(PMe₃)PhH is nearly 250 times slower than from Cp*Rh(PMe₃)PhH.     

Kinetics of circular DNA molecule digestion by restriction endonuclease Computation of kinetic constants from time dependence of fragment concentrations

A model for kinetics of circular substrate cleavage by restriction endonuclease was formulated. The aim of the analysis of the model was to extract kinetic constants for all target sites from time-dependence of fragment concentration in reaction products. That was proved to be possible for molecules with an odd number of fragments only. A symmetry of the molecules with an even number of fragment is the cause. A solution for molecules with an odd number of fragments was found and methods for dealing with the other molecules were suggested.Preliminary results were presented at VIIth CMEA Symposium Biophysics of Nucleic Acids and Proteins, Brno (Czechoslovakia) December 2–6, 1985.     

An evaluation of different enzymatic cleavage methods for recombinant fusion proteins, applied on des(1–3)insulin-like growth factor I

Different enzymatic methods for cleavage of recombinant fusion proteins were compared. To find an efficient cleavage method, five different fusion proteins were produced. The fusion proteins differed only in the linker region between the fusion partner and the desired product, human des(1–3)insulin-like growth factor I. A cleavage study was performed with enterokinase, plasmin, thrombin, urokinase, and recombinant H64A subtilisin. Significant cleavage was obtained using thrombin, H64A subtilisin, and enterokinase. Thrombin cleavage was studied on a larger scale and des(1–3)IGF-I was recovered at a final yield of 3 mg/L growth medium. Thrombin and enterokinase were also studied as immobilized proteases and they cleaved the fusion proteins with retained activity. To further improve thrombin cleavage, a continuous reactor was constructed, consisting of a closed system with a thrombin column and an ion exchange column in series. Here, the fusion protein circulated while free des(1–3)IGF-I was bound to the ion exchange column after release from the fusion protein. In the reactor, thrombin was as efficient as the free enzyme but gave a diminished rate of product degradation.     

The unique developmental program of the acoel flatworm, Neochildia fusca

Acoel embryos exhibit a unique form of development that some investigators argue is related to that found in polyclad turbellarians and coelomate spiralians, which display typical quartet spiral cleavage. We generated the first cell-lineage fate map for an acoel flatworm, Neochildia fusca, using modern intracellular lineage tracers to assess the degree of similarity between these distinct developmental programs. N. fusca develops via a "duet" cleavage pattern in which second cleavage occurs in a leiotropically oblique plane relative to the animal-vegetal axis. At the four-cell stage, the plane of first cleavage corresponds to the plane of bilateral symmetry. All remaining cleavages are symmetrical across the sagittal plane. No ectomesoderm is formed; the first three micromere duets generate only ectodermal derivatives. Endomesoderm, including the complex assemblage of circular, longitudinal, and oblique muscle fibers, as well as the peripheral and central parenchyma, is generated by both third duet macromeres. The cleavage pattern, fate map, and origins of mesoderm in N. fusca share little similarity to that exhibited by other spiralians, including the Platyhelminthes (e.g., polyclad turbellarians). These findings are considered in light of the possible evolutionary origins of the acoel duet cleavage program versus the more typical quartet spiral cleavage program. Finally, an understanding of the cell-lineage fate map allows us to interpret the results of earlier cell deletion studies examining the specification of cell fates within these embryos and reveals the existence of cell-cell inductive interactions in these embryos.     

A variant in the restriction endonuclease cleavage pattern of mitochondrial DNA in the domestic fowl, Gallus gallus domesticus

Summary. The cleavage patterns of mitochondrial DNAs (mtDNAs) were investigated from 15 lines of domestic fowls, Gallus gallus domesticus . using 11 restriction endonucleases. The cleavage patterns with 10 restriction endonucleases were identical in all the lines. A variant was found in a line of White Leghorn in the pattern with Mspl digestions. Cleavage patterns of the red jungle fowl, Gallus gallus gallus , were identical to the common patterns shown by the 14 lines of domestic fowls.     

Characterization of Sphingomonas paucimobilis SYK-6 genes involved in degradation of lignin-related compounds

Sphingomonas paucimobilis SYK-6 is able to grow on a wide variety of dimeric lignin compounds. These compounds are degraded via vanillate and syringate by a unique enzymatic system, composed of etherases, O demethylases, ring cleavage oxygenases and side chain cleaving enzymes. These unique and specific lignin modification enzymes are thought to be powerful tools for utilization of the most abundant aromatic biomass, lignin. Here, we focus on the genes and enzymes involved in β-aryl ether cleavage and biphenyl degradation. Two unique etherases are involved in the reductive cleavage of β-aryl ether. These two etherases have amino acid sequence similarity with the glutathione S-transferases, and use glutathione as a hydrogen donor. It was found that 5,5′-dehydrodivanillate, which is a typical lignin-related biphenyl structure, was transformed into 5-carboxyvanillate by the reaction sequence of O-demethylation, meta-ring cleavage, and hydrolysis, and the genes involved in the latter two reactions have been characterized. Vanillate and syringate are the most common intermediate metabolites in lignin catabolism. These compounds are initially O-demethylated and the resulting diol compounds, protocatechuate (PCA) and 3-O-methylgallate, respectively, are subjected to ring cleavage catalyzed by PCA 4,5-dioxygenase. The ring cleavage products generated are further degraded through the PCA 4,5-cleavage pathway. We have isolated and characterized genes for enzymes involved in this pathway. Disruption of a gene for 2-pyrone-4,6-dicarboxylate hydrolase (ligI) in this pathway suggested that an alternative route for 3-O-methylgallate degradation, in which ligI is not involved, would play a role in syringate catabolism. In this article, we describe the genetic and biochemical features of the S. paucimobilis SYK-6 genes involved in degradation of lignin-related compounds. A possible application of the SYK-6 lignin degradation system to produce a valuable chemical material is also described. Received 01 May 1999/ Accepted in revised form 29 July 1999     

A novel 1,2,4-triazole-based copper(II) complex: Synthesis, characterization, magnetic property and nuclease activity

A novel binuclear copper(II) complex [Cu₂L(μ-SO₄)](PF₆)₂ (1) (L = 3,5-bis (bis(pyridine-2-ylmethyl)amino)methyl)-4H-1,2,4-triazol-4-amine) has been synthesized and structurally characterized. X-ray structure shows that the two copper(II) atoms are bridged by one bidentate sulfate ion and the 1,2,4-triazole ring of L with Cu1?Cu2 distance of 4.404 Å. Each copper(II) center has a distorted trigonal-bipyramidal configuration. Variable-temperature magnetic susceptibility studies (2-300 K) indicate the existence of weak antiferromagnetic coupling between the copper(II) ions in complex 1. The interaction of complex 1 with calf thymus DNA (CT-DNA) has been studied by UV absorption, fluorescence spectroscopy, circular dichroism spectroscopy, viscosity and cyclic voltammetry. Furthermore, complex 1 was able to promote single and double strand DNA cleavage in both aerobic and anaerobic conditions, the pseudo-Michaelis-Menten kinetic parameters k_cat = 2.58 h⁻¹ and K_m = 1.2 × 10⁻⁴ M were obtained for 1. The hydrolytic cleavage of DNA by the complex was supported by the evidence from free radical quenching, anaerobic experiment, thiobarbituric acid-reactive substances (TBARS) assay.     

Plasmid-mediated degradation of o- and p-phthalate by Pseudomonas fluorescens

A Pseudomonas fluorescens strain SKP3 capable of utilizing both phthalic acid and terephthalic acid as sole source of carbon and energy was isolated by enrichment technique. Phthalic acid, terephthalic acid and protocatechuic acid were easily oxidized by both phthalate-grown and glucose-grown cells without a lag period. Phthalic acid is metabolized through the ortho cleavage pathway and terephthalic acid through the meta cleavage pathway and the enzymes of the two pathways are constitutive in nature. A large plasmid of approximately 140kb in size was found to be involved in the degradation of phthalates. The catabolic plasmid pSKL was transferable to different hosts.     

Generating recombinant C-terminal prion protein fragments of exact native sequence

Transmissibility and distinctive neuropathology are hallmark features of prion diseases differentiating them from other neurodegenerative disorders, with pathogenesis and transmission appearing closely linked to misfolded conformers (PrP(Sc)) of the ubiquitously expressed cellular form of the prion protein (PrP(C)). Given the apparent pathogenic primacy of misfolded PrP, the utilisation of peptides based on the prion protein has formed an integral approach for providing insights into misfolding pathways and pathogenic mechanisms. In parallel with studies employing prion peptides, similar approaches in other neurodegenerative disorders such as Alzheimer Disease, have demonstrated that differential processing of parent proteins and quite minor variations in the primary sequence of cognate peptides generated from the same constitutive processing (such as Aβ1-40 versus Aβ1-42 produced from γ-secretase activity) can be associated with very different pathogenic consequences. PrP(C) also undergoes constitutive α- or β-cleavage yielding C1 (residues 112-231 human sequence) or C2 (residues 90-231), respectively, with the full cell biological significance of such processing unresolved; however, it is noteworthy that in prion diseases, such as Creutzfeldt-Jakob disease (CJD) and murine models, the moderately extended C2 fragment predominates in the brain suggesting that the two cleavage events and the consequent C-terminal fragments may differ in their pathogenic significance. Accordingly, studies characterising biologically relevant peptides like C1 and C2, would be most valid if undertaken using peptides completely free of any inherent non-native sequence that arises as a by-product of commonly employed recombinant production techniques. To achieve this aim and thereby facilitate more representative biophysical and neurotoxicity studies, we adapted the combination of high fidelity Taq TA cloning with a SUMO-Hexa-His tag-type approach, incorporating the SUMO protease step. This technique consistently produced sufficient yields (～10 mg/L) of high purity peptides (>95%) equating to C1 and C2 of exact native primary sequence in the α-helical conformation suitable for biological and biophysical investigations.     

The relation of time to direction and equality of cleavage in Ilyanassa embryos

Summary

Cleavage inhibition experiments using cytochalasin B and hydrostatic pressure demonstrate the existence of a “clock mechanism” specifying cleavage time and form in Ilyanassa obsoleta embryos. Cytokinesis but not karyokinesis is inhibited during these treatments. When second cleavage is inhibited, the following cleavage occurs approximately on schedule with controls. Two micromeres are produced in this cleavage even though treated embryos consist of only two cells. When third cleavage is inhibited, the following micromere cleavage occurs in a counter clockwise direction, typical for the controls. Treatment with nocodazole, an antitubulin drug, inhibits both cytokinesis and karyokinesis but does not affect the cleavage clock mechanism. Treatment with 2,4-dinitrophenol stops both cleavage and the clock mechanism. These results indicate that the cleavage clock in Ilyanassa requires energy but does not depend on centrosomal behavior or on the form of previous cleavages. With regard to the production of micromeres the clock may involve an interaction between the aster-spindle complex and special regions of the animal pole cortex.